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FRAXE-associated mental retardation protein (FMR2) is an RNA-binding protein with high affinity for G-quartet RNA forming structure.

Bensaid M, Melko M, Bechara EG, Davidovic L, Berretta A, Catania MV, Gecz J, Lalli E, Bardoni B - Nucleic Acids Res. (2009)

Bottom Line: We show here that FMR2 is able to specifically bind the G-quartet-forming RNA structure with high affinity.Remarkably, in vivo, in the presence of FMR2, the ESE action of the G-quartet situated in mRNA of an alternatively spliced exon of a minigene or of the putative target FMR1 appears reduced.All together, our findings strongly suggest that FMR2 is an RNA-binding protein, which might be involved in alternative splicing regulation through an interaction with G-quartet RNA structure.

View Article: PubMed Central - PubMed

Affiliation: CNRS UMR 6097-Institut de Pharmacologie Moléculaire et Cellulaire, Valbonne, France.

ABSTRACT
FRAXE is a form of mild to moderate mental retardation due to the silencing of the FMR2 gene. The cellular function of FMR2 protein is presently unknown. By analogy with its homologue AF4, FMR2 was supposed to have a role in transcriptional regulation, but robust evidences supporting this hypothesis are lacking. We observed that FMR2 co-localizes with the splicing factor SC35 in nuclear speckles, the nuclear regions where splicing factors are concentrated, assembled and modified. Similarly to what was reported for splicing factors, blocking splicing or transcription leads to the accumulation of FMR2 in enlarged, rounded speckles. FMR2 is also localized in the nucleolus when splicing is blocked. We show here that FMR2 is able to specifically bind the G-quartet-forming RNA structure with high affinity. Remarkably, in vivo, in the presence of FMR2, the ESE action of the G-quartet situated in mRNA of an alternatively spliced exon of a minigene or of the putative target FMR1 appears reduced. Interestingly, FMR1 is silenced in the fragile X syndrome, another form of mental retardation. All together, our findings strongly suggest that FMR2 is an RNA-binding protein, which might be involved in alternative splicing regulation through an interaction with G-quartet RNA structure.

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FMR2 binds with high specificity the G-quartet RNA structure. (A) Sequence of the FBS purine-rich region encompassing the G-quartet forming structure inside the N19 RNA. The 35 nucleotides indicated in bold and underlined were deleted to generate the ΦBΣ Δ35 sequence. (B) Filter-binding assay using increasing amounts of full-length FMR2, FMRP, N-ter and C-ter proteins in the presence of K+ using the N19 RNA as labelled probe. (C) The same experience described in (B) was repeated in the presence of Na+ and in (D) in the presence of Li+. (E) Competition experiments in a nitrocellulose binding assay using the N19 unlabelled RNA as competitor and the unlabelled 3′UTR of PP2Ac RNA (N8) not containing any G-quartet forming structure as a negative control. (F) Filter-binding assay using an increasing amount of full-length FMR2, FMRP and C-ter proteins. The labelled probe is FBS RNA. (G) Filter-binding assay using an increasing amount of full-length FMR2 and C-ter proteins. The labelled probe is the ΦBΣ Δ35 RNA. Each point shows the mean of the results obtained in three independent experiments (see Supplementary Table 1 for details of each binding assay).
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Figure 5: FMR2 binds with high specificity the G-quartet RNA structure. (A) Sequence of the FBS purine-rich region encompassing the G-quartet forming structure inside the N19 RNA. The 35 nucleotides indicated in bold and underlined were deleted to generate the ΦBΣ Δ35 sequence. (B) Filter-binding assay using increasing amounts of full-length FMR2, FMRP, N-ter and C-ter proteins in the presence of K+ using the N19 RNA as labelled probe. (C) The same experience described in (B) was repeated in the presence of Na+ and in (D) in the presence of Li+. (E) Competition experiments in a nitrocellulose binding assay using the N19 unlabelled RNA as competitor and the unlabelled 3′UTR of PP2Ac RNA (N8) not containing any G-quartet forming structure as a negative control. (F) Filter-binding assay using an increasing amount of full-length FMR2, FMRP and C-ter proteins. The labelled probe is FBS RNA. (G) Filter-binding assay using an increasing amount of full-length FMR2 and C-ter proteins. The labelled probe is the ΦBΣ Δ35 RNA. Each point shows the mean of the results obtained in three independent experiments (see Supplementary Table 1 for details of each binding assay).

Mentions: G-quartets or quadruplexes are four stranded nucleic acid structures formed by stacking of planar layers of guanine tetrad units. In the tetrads, four guanines interact two by two in a cyclic Hoogsteen hydrogen bonding arrangement. G-quartet RNA folds in a 3D structure preferentially in the presence of K+ ions in comparison with Li+ and Na+ ions (18,29), as it was also shown for G-quartet RNA structure localized in FMR1 mRNA (18,22). To test our hypothesis, we generated recombinant N-ter, C-ter and full-length His-tagged FMR2 proteins and we performed a filter-binding assay with the N19 probe, using FMRP as a positive control. Surprisingly, in the presence of K+, full-length FMR2 and C-ter proteins were able to bind N19 as tightly as FMRP, whereas the N-terminal domain, in agreement with the results shown in Figure 4A and 4B, was not (Figure 5B). Additionally, in the presence of Na+, that partially destabilizes the G-quartet formation, FMR2 and C-ter bound to N19 to a much lower extent (Figure 5C), while in the presence of Li+ the binding of FMR2 proteins to N19 RNA was abolished (Figure 5D). To measure binding affinity we used competition assays that are more sensitive and allow for alleviation of the contribution of the non-specific binding properties of FMR2 (18,22). Therefore a constant concentration of labeled N19 RNA was incubated with a fixed amount of His-FMR2 and C-ter in the presence of increasing concentrations of unlabeled N19 RNA as a competitor. Binding of FMR2 and C-ter to the labeled N19 RNA was efficiently competed by the cold probe (Figure 5E). As little as 1 nM of competitor RNA is able to displace 50% of FMR2 from the G-quartet labeled probe, an affinity comparable to that of FMRP for G-quartet RNA structure (17,18,22). When we used the unlabelled N8 competitor RNA (corresponding to the 3′UTR of PP2Ac mRNA and not containing G-quartet RNA) (17) as a negative control, no displacement of the binding was observed for FMR2 (Figure 5E). Then we tested the ability of FMR2 and C-ter to bind the FBS (FMRP binding site) RNA, encompassing only the G-quartet forming structure inside the N19 RNA (Figure 5A) (nt 1557–1658 of the FMR1 mRNA coding region), FMRP, FMR2 and C-ter proteins were able to bind specifically to FBS (Figure 5F). Conversely, when we deleted the G-quartet structure generating the ΦBΣ Δ35 sequence [described in Figure 5A and in ref. (18)] full-length FMR2 and its C-terminal domain were unable to bind (Figure 5G). We can conclude that FMR2 binds G-quartet RNA specifically and with high affinity.Figure 5.


FRAXE-associated mental retardation protein (FMR2) is an RNA-binding protein with high affinity for G-quartet RNA forming structure.

Bensaid M, Melko M, Bechara EG, Davidovic L, Berretta A, Catania MV, Gecz J, Lalli E, Bardoni B - Nucleic Acids Res. (2009)

FMR2 binds with high specificity the G-quartet RNA structure. (A) Sequence of the FBS purine-rich region encompassing the G-quartet forming structure inside the N19 RNA. The 35 nucleotides indicated in bold and underlined were deleted to generate the ΦBΣ Δ35 sequence. (B) Filter-binding assay using increasing amounts of full-length FMR2, FMRP, N-ter and C-ter proteins in the presence of K+ using the N19 RNA as labelled probe. (C) The same experience described in (B) was repeated in the presence of Na+ and in (D) in the presence of Li+. (E) Competition experiments in a nitrocellulose binding assay using the N19 unlabelled RNA as competitor and the unlabelled 3′UTR of PP2Ac RNA (N8) not containing any G-quartet forming structure as a negative control. (F) Filter-binding assay using an increasing amount of full-length FMR2, FMRP and C-ter proteins. The labelled probe is FBS RNA. (G) Filter-binding assay using an increasing amount of full-length FMR2 and C-ter proteins. The labelled probe is the ΦBΣ Δ35 RNA. Each point shows the mean of the results obtained in three independent experiments (see Supplementary Table 1 for details of each binding assay).
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Figure 5: FMR2 binds with high specificity the G-quartet RNA structure. (A) Sequence of the FBS purine-rich region encompassing the G-quartet forming structure inside the N19 RNA. The 35 nucleotides indicated in bold and underlined were deleted to generate the ΦBΣ Δ35 sequence. (B) Filter-binding assay using increasing amounts of full-length FMR2, FMRP, N-ter and C-ter proteins in the presence of K+ using the N19 RNA as labelled probe. (C) The same experience described in (B) was repeated in the presence of Na+ and in (D) in the presence of Li+. (E) Competition experiments in a nitrocellulose binding assay using the N19 unlabelled RNA as competitor and the unlabelled 3′UTR of PP2Ac RNA (N8) not containing any G-quartet forming structure as a negative control. (F) Filter-binding assay using an increasing amount of full-length FMR2, FMRP and C-ter proteins. The labelled probe is FBS RNA. (G) Filter-binding assay using an increasing amount of full-length FMR2 and C-ter proteins. The labelled probe is the ΦBΣ Δ35 RNA. Each point shows the mean of the results obtained in three independent experiments (see Supplementary Table 1 for details of each binding assay).
Mentions: G-quartets or quadruplexes are four stranded nucleic acid structures formed by stacking of planar layers of guanine tetrad units. In the tetrads, four guanines interact two by two in a cyclic Hoogsteen hydrogen bonding arrangement. G-quartet RNA folds in a 3D structure preferentially in the presence of K+ ions in comparison with Li+ and Na+ ions (18,29), as it was also shown for G-quartet RNA structure localized in FMR1 mRNA (18,22). To test our hypothesis, we generated recombinant N-ter, C-ter and full-length His-tagged FMR2 proteins and we performed a filter-binding assay with the N19 probe, using FMRP as a positive control. Surprisingly, in the presence of K+, full-length FMR2 and C-ter proteins were able to bind N19 as tightly as FMRP, whereas the N-terminal domain, in agreement with the results shown in Figure 4A and 4B, was not (Figure 5B). Additionally, in the presence of Na+, that partially destabilizes the G-quartet formation, FMR2 and C-ter bound to N19 to a much lower extent (Figure 5C), while in the presence of Li+ the binding of FMR2 proteins to N19 RNA was abolished (Figure 5D). To measure binding affinity we used competition assays that are more sensitive and allow for alleviation of the contribution of the non-specific binding properties of FMR2 (18,22). Therefore a constant concentration of labeled N19 RNA was incubated with a fixed amount of His-FMR2 and C-ter in the presence of increasing concentrations of unlabeled N19 RNA as a competitor. Binding of FMR2 and C-ter to the labeled N19 RNA was efficiently competed by the cold probe (Figure 5E). As little as 1 nM of competitor RNA is able to displace 50% of FMR2 from the G-quartet labeled probe, an affinity comparable to that of FMRP for G-quartet RNA structure (17,18,22). When we used the unlabelled N8 competitor RNA (corresponding to the 3′UTR of PP2Ac mRNA and not containing G-quartet RNA) (17) as a negative control, no displacement of the binding was observed for FMR2 (Figure 5E). Then we tested the ability of FMR2 and C-ter to bind the FBS (FMRP binding site) RNA, encompassing only the G-quartet forming structure inside the N19 RNA (Figure 5A) (nt 1557–1658 of the FMR1 mRNA coding region), FMRP, FMR2 and C-ter proteins were able to bind specifically to FBS (Figure 5F). Conversely, when we deleted the G-quartet structure generating the ΦBΣ Δ35 sequence [described in Figure 5A and in ref. (18)] full-length FMR2 and its C-terminal domain were unable to bind (Figure 5G). We can conclude that FMR2 binds G-quartet RNA specifically and with high affinity.Figure 5.

Bottom Line: We show here that FMR2 is able to specifically bind the G-quartet-forming RNA structure with high affinity.Remarkably, in vivo, in the presence of FMR2, the ESE action of the G-quartet situated in mRNA of an alternatively spliced exon of a minigene or of the putative target FMR1 appears reduced.All together, our findings strongly suggest that FMR2 is an RNA-binding protein, which might be involved in alternative splicing regulation through an interaction with G-quartet RNA structure.

View Article: PubMed Central - PubMed

Affiliation: CNRS UMR 6097-Institut de Pharmacologie Moléculaire et Cellulaire, Valbonne, France.

ABSTRACT
FRAXE is a form of mild to moderate mental retardation due to the silencing of the FMR2 gene. The cellular function of FMR2 protein is presently unknown. By analogy with its homologue AF4, FMR2 was supposed to have a role in transcriptional regulation, but robust evidences supporting this hypothesis are lacking. We observed that FMR2 co-localizes with the splicing factor SC35 in nuclear speckles, the nuclear regions where splicing factors are concentrated, assembled and modified. Similarly to what was reported for splicing factors, blocking splicing or transcription leads to the accumulation of FMR2 in enlarged, rounded speckles. FMR2 is also localized in the nucleolus when splicing is blocked. We show here that FMR2 is able to specifically bind the G-quartet-forming RNA structure with high affinity. Remarkably, in vivo, in the presence of FMR2, the ESE action of the G-quartet situated in mRNA of an alternatively spliced exon of a minigene or of the putative target FMR1 appears reduced. Interestingly, FMR1 is silenced in the fragile X syndrome, another form of mental retardation. All together, our findings strongly suggest that FMR2 is an RNA-binding protein, which might be involved in alternative splicing regulation through an interaction with G-quartet RNA structure.

Show MeSH
Related in: MedlinePlus